Bifunctional molecules, commonly referred to as crosslinkers, are used to connect two molecules together. Bifunctional molecules can contain homo or hetero-bifunctionality. The reversibility of disulfide bond formation makes them useful tools for the transient attachment of two molecules. Disulfides have been used to attach a bioactive compound and another compound (Thorpe 1987). Reduction of the disulfide bond releases the bioactive compound. Disulfide bonds may also be used in the formation of polymers (Kishore et al 1993).
There are many commercially available reagents for the linkage of two molecules by a disulfide bond. Additionally there are bifunctional reagents that have a disulfide bond present. Typically, these reagents are based on 3-mercaptopropionic acid, i.e. dithiobispropionate. However, the rate at which these bonds are broken under physiological conditions is slow. For example, the half life of a disulfide derived from dithiobispropionimidate, an analog of 3-mercaptopropionic acid, is 27 h in vivo (Arpicco et al. 1997). A stable disulfide bond is often desirable, for example when purification of linked molecules or long circulation in vivo is needed. For this reason, attempts have been made to make the disulfide less susceptible to cleavage.
It has been demonstrated that both stability, measured as reduction potential, and rate, measured as rate constants, of disulfide reduction are both related to the acidity of the thiols which constitute the disulfide. Additional factors that may affect the rate of reduction are steric interactions and intramolecular disulfide cleavage. Looking at the difference in the rates for the reactions RSH+R′SSR′→RSSR′+R′SH and RSH+R″SSR″→RSSR″+R″SH, it has been demonstrated that log k″/k′=β(pKaR′-pKaR″), where k′ and k″ are the rate constant for the reactions with R′SSR′ and R″SSR″ respectively, pKaR′ and pKaR″ are the acidities of the thiol groups R′SH and R″SH, and β is a constant determined empirically to be 0.72. From this equation, one would predict that the reduction of a disulfide composed from relatively acidic thiols would be reduced more quickly than one composed of less acidic thiols. In support of this observation, it has been demonstrated that the disulfides cystine (pKa 8.3) and cystamine (pKa 8.2) are reduced 3-15 times faster than oxidized glutathione (pKa 8.9) (Bulaj et al. 1998).
It has been demonstrated that both stability (thermodynamics), measured as reduction potential (Keire 1992), and rate (kinetics), measured as rate constants, of disulfide reduction are both related to the acidity of the thiols which constitute the disulfide (Szajewski et al. 1980). The increase in acidity of a thiol is dependent upon one or more of the following structural factors: the presence of electron withdrawing groups which stabilize the thiolate through sigma and pi bonds (inductive effect), the presence of electron withdrawing groups that stabilize the thiolate through space or solvent (field effects), pi bonds which allow the negative charge to be placed on other atoms (resonance stabilization), and hydrogen bond donating groups within the molecule that can interact internally with the thiolate. For example, cysteine has an amino group two atoms from the thiol, which is more electron withdrawing than the amide nitrogen that is two atoms from the thiol in glutathione. As a consequence of this difference in electron withdrawing groups, the thiol of cysteine is 0.6 pK units more acidic than glutathione, and as mentioned previously, cystine is reduced 3-15 times faster than oxidized glutathione. Another example of a relatively acidic thiol is 5-thio-2-nitrobenzoic acid, pKa 5. Its acidity is due to resonance stabilization and inductive effects. Its disulfide is rapidly reduced by all standard alkyl thiols and its colored thiolate makes it a convenient assay for thiol concentration.